MR-guided radiotherapy (MRgRT) provides superior soft-tissue contrast over CT-based image guidance. We collected and analyzed daily pre-treatment (PRE) and real-time motion-monitoring (MM) MR images of patients receiving prostate radiotherapy to assess interfractional and intrafractional variability of prostate using two localization methods: pelvic bony anatomy (bone) and prostate during online adaptive radiotherapy (ART). PRE and MM MRIs for the first five fractions of twenty prostate cancer patients who received definitive MRgRT with 1.5T MRI were collected. Using MIM software, rigid registration between PRE MRI and planning CT images based on pelvic bony anatomy and prostate reproduced bone localization and online ART, respectively. To determine interfractional setup margin (SM), prostate was delineated on all PRE MRIs registered after bone and prostate localizations by a radiation oncologist, and centroid values of prostate contours between planning CT and PRE MRIs were compared. To determine interobserver variability, another radiation oncologist, a medical physicist, and a radiotherapist contoured prostate for both localization methods. For internal margin (IM) assessment, we used MM MRIs of the five patients who had all three sets of coronal, sagittal, and axial cine images and determined the maximum contour displacement using in-house MATLAB-based software converting binary image files to 2D cine images with a superimposed grid of 1 mm spacing. A total of 100 PRE and 25 MM MRIs were analyzed. Four hundred prostate contours were delineated on MR images registered with planning CT based on both bony anatomy and prostate. After bone localization, SM was 0.57±0.42 mm in left-right (LR), 2.45±1.98 mm in anterior-posterior (AP), and 2.28±2.08 mm in superior-inferior (SI) directions, and IO was 1.06±0.58 mm in LR, 2.32±1.08 mm in AP, and 3.30±1.85 mm in SI directions. After prostate localization, SM was 0.76±0.57 mm in LR, 1.89±1.60 mm in AP, and 2.2±1.79 mm in SI directions, and IO was 1.11±0.55 mm in LR, 2.13±1.07 mm in AP, and 3.53±1.65 mm in SI directions. Average IM was 2.12±0.86 mm, 2.24±1.07 mm, and 2.84±0.88 mm in LR, AP, and SI directions, respectively. Using daily MRIs from MRgRT, we showed that movements in the SI direction were the largest source of variability in prostate definitive RT. In addition, interobserver variability was a non-negligible source of margin. Optimal PTV margin should also consider internal margin, especially in the SI direction.